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U1 and U4 snRNP results in the B* complex, which forms the catalytic core of the spliceosome [Staley
and Guthrie, 1998, Liu
et al.
, 2006]. The B-complex, composed of U4/U6 U5 tri-snRNP in close contact
with the U2 snRNP, performs the first catalytic step of splicing by nucleophilic attack of the branch point
adenosine to the phosphate ester bond of the 5'ss, and is subsequently converted into the C-complex.
C-complex
The C-complex contains U2, U5 and U6 snRNA at a stage subsequent to the first catalytical step since
splicing intermediates can be found in this complex. The conformation is centered around the protein
Prp8, which is thought to serve as a “surgery table”, connecting the already free 5'ss and fixating the
3'ss such that the second transesterification can open the downstream intron-exon junction [Turner
et
al.
, 2004]. With the ligation of the free exon ends, the intron and its bound snRNPs are released as
lariat complex. Subsequently, bonds between the U2, U6 and U5 snRNA are broken, involving the
helicase Prp43, and the U5 snRNP is dissociating into its components, being available for a new cycle of
spliceosome assembly [Makarov
et al.
, 2002]. Since Prp43 can be found in the 17S U2 complex [Will
et al.
, 2002], which forms during early A-complex assembly, it is conceivable that this protein is present
in several stages of the spliceosome assembly pathway.
Additional factors support the recycling process, for example, Prp24, which reanneals the U4 and U6
snRNAs and allows regeneration of the U4/U6 snRNP duplex [Gottschalk
et al.
, 2001]. Two important
helicases, Prp16 and Prp22, impose kinetic proofreading activity and can subject suboptimal splicing
substrates into a proposed discard pathway [Burgess and Guthrie, 1993; Villa and Guthrie, 2005; Mayas
et al.
, 2006]. It is important to note that the catalyzing function of the spliceosome can experimentally be
reduced to its RNA parts, thus, making it functioning as a ribozyme [Valadkhan
et al.
, 2007]. However,
the protein scaffold is necessary to form the structural environment (RNA conformations) necessary
to enable the splicing reaction. Moreover, the participating proteins establish important links to other
cellular processes, for example, transcription or nuclear export.
These known experimental results were taken as base to extract reactions applicable for designing the
PN model. This often requires to consult several literature reports to model reactions, which are only
vaguely or contradictory described. Depending on available data, reactions and their participating factors
were summarized or abstracted. We introduce a mnemonic labeling for reactions (
e.g.
,“ bdg” for binding
“ matur” for maturation, “ ass” for assignment). All reactions used for the model are summarized in
Supplementary Table S1.
METHODS AND DEFINITIONS
Definition of Petri nets
We modeled the spliceosomal assembly network as a P/T net (see Box 1). Places correspond to
biological objects (
e.g.
, RNA regions, protein factors, protein complexes etc.), whereas transitions
correspond to processes, which act upon objects (
e.g.
, protein interaction, phosphorylation reactions,
proteinmRNA binding etc.). The direction of arcs defines pre-places (pre-transitions) and post-places
(post-transitions). Tokens represent movable objects. They are used to model the equivalent of signal or
mass flow units as a number of molecules (
e.g.
, mole) and are symbolized by black dots on places. The
maximal number of tokens that a place can hold is defined by its
capacity
. The distribution of tokens
over all places is called a
marking
.
Each marking defines a certain state of the system.
Transitions
without pre-places are called input transitions and represent sources.
Transitions without post-places
